Additional Supporting Information may be found in the online version of this article.

HEP_23848_sm_SuppFig-1.tif818KSupporting Figure 1: SULF2 increases Wnt/β-catenin signaling in PLC/PRF/5 cells PLC/PRF/5 cells were transfected with TOPFLASH reporter construct and either a SULF2-expressing construct or empty vector. After serum-starvation, cells were treated with 5 ng/ml Wnt3a ligand and TOPFLASH luciferase activity was measured after 6 hours (A) or 24 hours (B). SULF2 enhanced Wnt3a-induced luciferase activity as early as 6 hours and the effect was sustained over 24 hours (p<0.0271).
HEP_23848_sm_SuppFig-2.tif122KSupporting Figure 2: Expression of SULF2 up-regulates the canonical Wnt signaling pathway Western immunoblotting was performed on whole cell lysates from Hep3B Vector (control), Hep3B SULF2-L (low SULF2 expression) and Hep3B SULF2-H (high SULF2 expression) cells using antibodies against phospho-GSK3β, total GSK3β, β-catenin, phospho-β-catenin and actin (loading control). Increased SULF2 expression resulted in increased phosphorylation of GSK3β and a concomitant decrease in phospho-β-catenin, which is then ubiquitinated and degraded.
HEP_23848_sm_SuppFig-3.tif128KSupporting Figure 3: SULF2 increases membrane and cytoplasmic β-catenin in HCC cells Hep3B cells were transiently transfected with either SULF2-expressing plasmid construct or an empty vector. SULF2 was associated with both increased membrane and cytoplasmic β-catenin as seen by immunofluorescence.

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